Petroleum prospecting



a States ate This invention relates to a biological method forprospecting for petroleum deposits which involves the use of serologicaltechniques to obtain indicia as to the proximity of an undergroundpetroleum reservoir.

Methods of prospecting for petroleum have been pro posed heretoforewherein samples of earth from selected sites have been analyzed invarious ways to indicate Whether hydrocarbons or products resulting frombac terial decomposition of hydrocarbons are present. It has beenpostulated that hydrocarbon gases migrate through the earth fromsubterranean petroleum reservoirs and hence are present in the earthnear the surface in the vicinity of petroleum deposits. It has proveddifficult, however, to make reliable determinations of hydrocarboncontent of soil samples due to the minute amount that may be present.

In soil subject to emanation of hydrocarbons from a petroleum deposit,microorganisms are generally present which consume or feed upon thehydrocarbon material. Accordingly it has been proposed to subject soilsamples in the laboratory to an atmosphere containing a hydrocarbon gas,e.g. ethane, under growth conditions to allow the hydrocarbon-consumingmicroorganisms to multiply if any are present, and thereafter determinethe extent of growth or the rate of consumption of the hydrocarbon.These values have been taken as indicia of the proximity of petroleumdeposits with respect to the sampling location. Such procedures,however, have the drawback of being tedious and time-consuming, and theprogress that can be made in running a prospecting survey over a widearea by such methods is slow.

The present invention provides an improved method of petroleumprospecting, whereby the presence of a hydrocarbon-consumingmicroorganism in soil samples can be rapidly determined. The methodutilizes serological techniques for determining the presence or absenceof a selected type of hydrocarbon-consuming microorganism and also theconcentration thereof in the sample. The method is so readily performedthat it is possible for a person to make several hundred soil tests in asingle day.

In practicing the invention an antiserum is prepared which is specifictoward any particular hydrocarbonconsuming microorganism which is knownto be prevalent in soil subject to seepage of petroleum hydrocarbongases. It is preferred that the antiserum be specific for Mycobacteriumparafiinicum, since this type of bacteria is practically universallypresent in soil which is subject to continuous contact by the paraffinhydrocarbon gases. However, the antiserum can be specific for any othermicroorganisms that can feed on the gaseous paraffin hydrocarbons, forexample, Agrobacterium ethanicus, Bacterium Iridium, Methanomonasmethanica and Bacillus ethanicus. Soil samples are collected fromselected locations and a small amount of each sample is allowed toincubate for a time with a small volume of the antiserum. The incubatedantiserum is then filtered from the soil and serologically tested atvarious dilutions with a culture of the microorganism which is anantigen for the antibody of the serum. If no reaction is obtained, thismeans that the soil had removed the antibody from the serum and hencethat the soil contained the hydrocarbon-consuming microorganism. On theother hand, if heavy coagulation is observed, the absence of themicroorganism in the soil sample is indicated. The test can be doneunder specific conditions that can be compared with a standardizedcontrol test and the concentration of the microorganism thereby can beascertained. Hence an area can be mapped in terms of the quantities ofthe hydrocarbon-consuming microorganism present in the soil so as toindicate Where a petroleum deposit likely is to be found.

The antiserum for use in practicing the invention is prepared byconventional biological techniques utilizing any suitable animal such asrabbit, hamster, chicken, horse or monkey. A culture of themicroorganism, preferably Mycobacterium parafiinicum, is grown in ahydrocarbon-containing atmosphere which hydrocarbon preferably isethane, and the microorganism is rendered avirulent in any suitablemanner such as by heat killing, formalizing, phenolizing or attenuation.The animal is then injected with the avirulent microorganism over aperiod of several days. Preferably the first injection is subcutaneousand is followed by three or four intravenous injections on successivedays. On about the sixth day the animals blood will contain the desiredantibodies and a quantity of it can be withdrawn. The blood is allowedto clot and stand at room temperature for at least three hours, and theserum is then decanted. It may be desirable to inactivate the serum at56 C. for 45 minutes in order to destroy complement since the bacterialcells may be lysed in some instances. The serum can be stored underrefrigeration at 0 C. The titer of the antiserum can be determined bydilution tests in conventional manner.

In treating the soil sample only small amounts of the soil, e.g., 0.1gram, and of the antiserum, e.g., 2-5 ml., need be used. The antiserumwash and the soil sample are mixed and allowed to incubate for at leastone hour at room temperature. The mixture may, if desired, be storedovernight at 4 C. The incubated antiserum is then filtered from the soiland is ready for serological testing.

Any of the known serological tests can be used for determining thepresence, absence or the amount of antibody remaining in the incubatedantiserum. Tests known as neutralization, agglutination, precipitin andcomplement fixation can be used, with neutralization being the preferredprocedure. In general these involve admixing the antiserum with a normalsaline preparation of the hydrocarbon-consuming bacteria (eithervirulent or avirulent) in a series of standard dilutions and observingthe reaction. In cases Where quantitative values are de sired,comparisons can be made with standard control tests in which a knownnumber of bacteria are reacted with various dilutions of the antiserum.Description of the various serological methods used in microbiologicalwork are available in various texts, for example, by W. C. Boyd,Fundamentals of Immunology, 3rd edition (1956), and hence need not bedescribed in detail here.

The invention can also be practiced by subjecting each soil sample to aplurality of tests carried out in the above-described manner but usingseveral different antisera corresponding to different species ofhydrocarbonconsuming microorganisms. This is generally not necessary,however, in view of the wide prevalence of Mycobacterium parafiinicum inhydrocarbon-containing soils. Hence tests made only with antiserumprepared by means of this type of microbe are sufiiciently reliable toindicate whether a soil sample has been continuously subjected to ahydrocarbon-containing atmosphere and thus whether petroleum is probablydeposited near the sampling site.

We claim:

1. Method of biological prospecting for petroleum deposits whichcomprises collecting soil samples from se- V 3 l lected locations;incubating each soil sample with an antipetroleum deposit with respectto the sampling location. serum, said antiserum having been prepared byinoculat- 2. Method according to claim 1 wherein said microing an animalwith an avirulent microorganism of a organism is Mycobacteriumparaflinicum. type capable of consuming gaseous petroleum hydrocar- 3.Method according to claim 1 wherein said serologibon to form antibodiesin the animals blood stream and 5 cally testing involves ascertainingthe concentration of separating the antiserum from the blood; separatingthe aid microorganism in the soil soil sample from the incubatedantiserum; and serologically testing the incubated antiserum With saidmicroorganism to ascertain Whether such microorganism Was y Fundamentalsof gy, 3rd 1956, present in the soil as an indicium of the proximity ofa 10 Intefscience -9 New York, PP- 174475 and References Cited in thefile of this patent

1. METHOD OF BIOLOGICAL PROSPECTING FOR PETROLEUM DEPOSITS WHICHCOMPRISES COLLECTING SOIL SAMPLES FROM SELECTED LOCATIONS; INCUBATINGEACH SOIL SAMPLE WITH AN ANTISERUM, SAID ANTISERUM HAVING BEEN PREPAREDBY INOCULATING AN ANIMAL WITH AN AVIRULENT MICROORGANISM OF A TYPECAPABLE OF CONSUMING GASEOUS PETROLEUM HYDROCARBON TO FORM ANTIBODIES INTHE ANIMAL''S BLOOD STREAM AND SEPARATING THE ANTISERUM FROM THE BLOOD;SEPARATING THE SOIL SAMPLE FROM THE INCUBATED ANTISERUM; ANDSEROLOGICALLY TESTING THE INCUBATED ANTISERUM WITH SAID MICROORGANISM TOASCERTAIN WHETHER SUCH MICROORGANISM WAS PRESENT IN THE SOIL AS ANINDICIUM OF THE PROXIMITY OF A PETROLEUM DEPOSIT WITH RESPECT TO THESAMPLING LOCATION.